Fusion protein comprising granzyme b and use thereof

ABSTRACT

A fusion protein including granzyme B, a cell penetrating peptide, a cleavage site, and a targeting moiety, a composition for cell membrane penetration comprising the fusion protein, and an anticancer composition comprising the fusion protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2014-0060005 filed on May 19, 2014 in the Korean IntellectualProperty Office, the entire disclosure of which is hereby incorporatedby reference.

INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: One 106,123 byte ASCII (Text) file named “720524 ST25.TXT”created May 7, 2015.

BACKGROUND OF THE INVENTION

1. Field

Provided are a fusion protein including granzyme B, a cell penetratingpeptide, a cleavage site of a peptidase or protease, and a targetingmoiety, a pharmaceutical composition for cell membrane penetrationincluding the fusion protein, and an anticancer composition includingthe fusion protein.

2. Description of the Related Art

It has been generally required in various clinical circumstances tospecifically kill a cancer cell. A number of intracellular signaltransduction pathways are associated with cell death and cell survival,and thus cell death and cell survival can be controlled by controllingpathways associated with cell death and survival. The pathways can besuccessfully controlled by the successful delivery of a substance thatrestricts or blocks the pathway. A representative example of the signaltransduction pathways is an apoptosis pathway. Thus, the elementsrelated to the apoptosis pathway are useful as a target to specificallydestroy a particular cell.

Granzyme B (GrB), a serine protease, is found in cytotoxic T lymphocytes(CTL) and natural killer (NK) cells. Granzyme B relates to apoptosiswhich is induced in a target cell when it is exposed to the contents oflysosome-type cytoplasmic granules (or lytic granules). Cytotoxic Tlymphocyte granules include a series of serine proteases includingperforin, a pore-forming protein, and granzymes.

In a lymphocyte-mediated cell lysis, perforin has been known to insertitself into a plasma membrane of a target cell and polymerize, forming apore, thereby mediating the access of granzyme B into the cytoplasm ofthe target cell. Once granzyme B is introduced inside of a cell, itdirectly activates a caspase and induces rapid DNA destruction, therebyinducing cellular apoptosis.

Granzymes are structurally related to each other, but have differentsubstrate preferences. Among the various granzymes, granzyme B has aparticular activity to cleave a procaspase after an aspartic acidresidue. and causes the maturation (i.e., activation) of the procapsaseinto caspase-3, thereby exhibiting a high cytotoxicity to a target cellinducing cellular apoptosis. Granzyme B directly activates caspasesunder certain conditions, directly damaging downstream caspasesubstrates, to trigger apoptosis.

Recently, various technologies to introduce macromolecules such asproteins into cells have been developed and are central to certain newtherapies. However, exact targeting of a cell or tissue is difficultusing existing technologies. In an effort to solve this problem, therehave been many studies conducted regarding the cell membrane penetrationof therapeutic proteins.

It has recently been found that a TAT protein of HIV-1 which is added toa cell culture medium can be delivered inside of a cell, and thus, theprotein has been known as a protein transduction domain (PTD). PTD canbe transferred into a cell together with other peptide or proteins whichare fused thereto. Thus, there have been various attempts to transfertherapeutic drugs, peptides, and proteins into a cell using the PTD.

It is required to develop a technology for intracellular delivery of acytotoxic substance such as granzyme B using a protein transductiondomain.

BRIEF SUMMARY OF THE INVENTION

Provided is a fusion protein including granzyme B, a cell penetratingpeptide (CPP), a cleavage site of a peptidase or protease, and atargeting moiety. The cell penetrating peptide may include a hydrophobicpeptide and a basic peptide.

Another embodiment provides a pharmaceutical composition including thefusion protein, and a carrier.

Another embodiment provides a composition for cell membrane penetrationof granzyme B including the fusion protein.

Another embodiment provides an anticancer composition including thefusion protein.

Another embodiment provides a method for intracellular delivery ofgranzyme B comprising administering the fusion protein to a subject inneed thereof.

Another embodiment provides a method of preventing and/or treating acancer including administering the fusion protein to a subject in needthereof.

Related compositions and methods also are provided in the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating a construct of various embodiments ofthe fusion protein described herein.

FIG. 2 illustrates immunoblotting assay results showing the expressionof a fusion protein.

FIG. 3 is a graph showing cell viability of human colon cancer cell lineHCT116 (ATCC) when treated with a fusion protein including granzyme B.

FIG. 4 is a graph showing cell viability of human colon cancer cell lineRKO (ATCC) when treated with a fusion protein including granzyme B.

FIGS. 5A to 5G provide tables displaying nucleotide sequences of variousDARPins.

DETAILED DESCRIPTION OF THE INVENTION

To more effectively treat a cancer, a fusion protein for intracellulardelivery of granzyme B is provided, wherein granzyme B exhibiting ananticancer effect and a cell penetrating peptide (CPP) for successfullydelivering granzyme B into a cancer cell are fused (conjugated), and amore effective anticancer effect can be achieved by delivering thedesigned fusion protein into a cancer cell.

An embodiment provides a fusion protein comprising (1) granzyme B, (2) acell penetrating peptide (CPP), (3) a cleavage site of a peptidase orprotease, and (4) a targeting moiety.

Granzyme B, which is a serine protease, is expressed in cytotoxic Tlymphocytes (CTL) and natural killer (NK) cell, and plays an importantrole in inducing apoptosis of a target cell in cell-mediate immuneresponse. Granzyme B has strong cytotoxicity. It is inactivated in anormal cell, but specifically activated in a cancer cell or avirus-infected cell by being cleaved at a specific site by a specificenzyme such as cathepsin, which is rich in a cancer cell orvirus-infected cell such cell, to exhibit cytotoxicity. Therefore,granzyme B is harmless to a normal cell but has specific and strongcytotoxicity to a cancer cell, and thus, it is advantageous for moreeffective cancer therapy with safety to a normal cell.

Granzyme B (EC number: 3.4.21.79) may be from a mammal, for example aprimate (such as human, a monkey, etc.), a rodent (such as a mouse, rat,etc.), and the like. For example, granzyme B may be selected from thegroup consisting of a human granzyme B comprising the amino acidsequence of Accession number NP_(—)004122 or an amino acid sequenceencoded by the nucleotide sequence (mRNA) of Accession numberNM_(—)004131, and a mouse granzyme B comprising the amino acid sequenceof Accession number NP_(—)038570, or a combination thereof; but not belimited thereto.

In the fusion protein, the granzyme B may be a full-length granzyme B ora fragment of granzyme B. The fragment of granzyme B may comprise anactive region (e.g.,“IIGGHEAKPHSRPYMAYLMIWDQKSLKRCGGFLIQDDFVLTAAHCWGSSINVTLGAHNIKEQEPTQQFIPVKRPIPHPAYNPKNFSNDIMLLQLERKAKRTRAVQPLRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDSTIELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIVSYGRNNGMPPRACTKVSSFVHWIKKTMKRH”: SEQ ID NO: 11) andcleavage sequence (or a cleavage inducing sequence; for example, acathepsin cleavage sequence or an auto-cleavage (self-cleavage) sequencecleaved by auto-activation (self-activation) of granzyme B). Thegranzyme B is activated by cleavage by a dipeptidyl peptidase I (DPPI),which is a representative cysteine-based protease found in ribosomes, orauto-activated (self-activated). The dipeptidyl peptidase I may becathepsin C or cathepsin H. Cathepsin C can exhibit its activity, unless(i) the amino group at the N-terminus of granzyme B is blocked, (ii) anyamino acid residue of the cleavage site is proline, (iii) the amino acidresidue at the N-terminus of granzyme B is lysine or arginine, or (iv)the structure of a peptide of protein disturbs cleavage (e.g., by afolding of a peptide or protein preventing the cleavage site from beingexposed). The auto-activation (self-activation) of granzyme B may beinduced by presence or artificial insertion of an auto-activationinducing sequence (e.g., “IEPD (SEQ ID NO: 36”). For example, thecleavage sequence in the fragment of granzyme B may be a cathepsincleavage site (for example, a cleavage site of Dipeptidyl peptidase I)of granzyme B, such as the native granzyme B DPPI cleavage site (e.g.,“GE”). Also, the cleavage sequence may be provided by a peptidecomprising an amino acid sequence (auto-cleavage sequence; e.g., IEPD(SEQ ID NO: 36), etc.) which is artificially linked (e.g., a non-nativepeptide) to an active region of granzyme B and induces anauto-activation and cleavage of granzyme B itself whereby the cleavagesequence (e.g., IEPD) is removed. In order to be cleaved by an enzymesuch as cathepsin, the cleavage sequence (peptide) may be exposed toN-terminus. Therefore, in the fragment of granzyme B, the cleavagesequence may be linked to N-terminus of the active region of granzyme B.

In an embodiment, the fragment of granzyme B may comprise:

1) an active region of granzyme B and

2) at least one cleavage sequence, for example, at least one cleavagesequence of dipeptidyl peptidase I (cleavage site recognized bydipeptidyl peptidase I) or at least one auto-cleavage sequence, whereinthe cleavage sequence comprises or consists essentially of about 2 toabout 20 amino acids, about 2 to about 15 amino acids, or about 2 toabout 10 amino acids, comprising contiguous amino acids ‘GE’, ‘IEPD’(SEQ ID NO: 36), or a combination thereof, wherein the cleavage site islinked to N-terminus of the active region of granzyme B.

As used herein, the term “cleavage sequence” in the fragment of granzymeB is used to be distinguished from the term “cleavage site of apeptidase or protease” positioned between a cell penetrating peptide(CPP) and a targeting moiety in a fusion protein described herein.

In order to be recognized by an enzyme such as cathepsin and activated,granzyme B may preferably have an exposed N-terminus. More specifically,the N-terminus of the cleavage sequence of granzyme B or the fragment ofgranzyme B should be exposed. Therefore, in the fusion protein, granzymeB or the fragment of granzyme B comprising the cleavage sequence, may bepositioned at N-terminal part of the fusion protein.

The cell penetrating peptide may be any peptide capable of penetratingcell membrane, and for example, at least one selected from the groupconsisting of a membrane-translocation sequence (MTS) or a fragmentthereof (wherein the fragment comprises 5 or more contiguous amino acidresidues in the membrane-translocation sequence), a macromoleculeintracellular transduction domain (MTD), TAT peptide (e.g., YGRKKRRQRRR;SEQ ID NO: 24), RKKRRQRRR (SEQ ID NO: 25)), MTD103 (LALPVLLLA; SEQ IDNO: 26), TP10 (AGYLLGKINLKALAALAKKIL; SEQ ID NO: 27), Penetratin(RQIKIWFQNRRMKWKK; SEQ ID NO: 28), MAP (model amphipathic peptide; e.g.,KLALKLALKALKAALKLA (SEQ ID NO: 29)), a cell penetrating fusion peptidecomprising a hydrophobic peptide and a basic peptide, or any combinationthereof.

For example, the membrane-translocation sequence may comprise or consistessentially of the amino acid sequence of AAVALLPAVLLALLAP (SEQ ID NO:12), but not be limited thereto. The fragment thereof may be a peptidecomprising or consisting essentially of about 7 to about 16 contiguousamino acid residues of the amino acid sequence of SEQ ID NO: 12, and forexample, a peptide comprising or consisting essentially of AAVALLP (SEQID NO: 13) or AVLLALLAP (SEQ ID NO: 14), but not be limited thereto.

The cell penetrating fusion peptide of a hydrophobic peptide and a basicpeptide may comprise or consist essentially of:

a hydrophobic peptide comprising a total of about 5 to about 100 aminoacids, about 5 to about 50 amino acids, about 5 to about 40 amino acids,or about 6 to 30 amino acids, wherein the hydrophobic peptide compriseshydrophobic amino acids at the ratio of about 60% or more, about 70% ormore, about 80% or more, or about 90% or more, for example, about 60 toabout 100%, about 70 to about 100%, about 80 to about 100%, or about 90to about 100%, based on the number of the total amino acids in thehydrophobic peptide; and

a basic peptide comprising a basic peptide unit comprising about 1 toabout 6 basic amino acids or a repeat comprising about 2 to about 6basic peptide units.

In the cell penetrating fusion peptide, the hydrophobic peptide maycomprise a total of about 5 to about 100 amino acids, about 5 to about50 amino acids, about 5 to about 40 amino acids, or about 6 to about 30amino acids, and comprise a hydrophobic amino acid at the ratio of about60% or more, about 70% or more, about 80% or more, or about 90% or more,for example, about 60 to about 100%, about 70 to about 100%, about 80 toabout 100%, or about 90 to about 100%, based on the number of the totalamino acids in the hydrophobic peptide. The hydrophobic amino acid maybe at least one selected from the group consisting of glycine, alanine,valine, leucine, isoleucine, methionine, proline, tryptophan,phenylalanine, and the like, or any combination thereof. When thehydrophobic peptide includes one kind of hydrophobic amino acid, thehydrophobic amino acid may be included once or repeatedly. In addition,in some embodiments the hydrophobic peptide may not comprise any basicamino acid.

In an embodiment, the hydrophobic peptide may be at least one selectedfrom the group consisting of a membrane-translocation sequence (MTS,e.g., AAVALLPAVLLALLAP; SEQ ID NO: 12), a fragment of themembrane-translocation sequence (for example, a peptide fragmentincluding about 7 to about 16 consecutive amino acids within the aminoacid sequence of SEQ ID NO: 1; e.g., AAVALLP (SEQ ID NO: 13), AVLLALLAP(SEQ ID NO: 14), etc.), and the like or any combination thereof.

The basic peptide may comprise or consist essentially of a basic peptideunit comprising about 1 to about 6 basic amino acids, or a repeatcomprising about 2 to about 6 basic peptide units. When the basicpeptide includes 2 or more basic amino acids, the basic amino acids arethe same with or different from one another. If the basic peptidecomprises 7 or more amino acids, the basic peptide acts as a cellpenetrating peptide in itself, thereby transferring a protein (e.g.,granzyme B) to an endosome, which can lead to the degradation of theprotein (e.g., granzyme B). Therefore, it may be advantageous that thebasic peptide includes 6 or less amino acid. The basic peptide may playa role to induce transfer of granzyme B into a nucleus.

The basic amino acid may be at least one selected from the groupconsisting of lysine, arginine, histidine, and the like, or anycombination thereof. When the basic peptide includes two or more basicamino acids, each of the basic amino acids may be independently selectedfrom the group consisting of lysine, arginine, histidine, and the like.When the basic peptide includes one kind of basic amino acid, the basicamino acid may be included once or repeatedly. When the basic peptidecomprises about 2 to about 6 basic peptide units, the basic peptideunits may be comprise the same amino acid sequence with or differentbasic peptide units from one another.

In a particular embodiment, the basic peptide unit may comprise lysine(K), arginine (R), or a combination thereof, in about 1 to about 6 aminoacids in length. For example, the basic peptide unit may be at least oneselected from the group consisting of KKKRK (SEQ ID NO: 15), KKKR (SEQID NO: 16), RKRK (SEQ ID NO: 17), RKRKRK (SEQ ID NO: 18), KKKKK (SEQ IDNO: 19), KKKKKR (SEQ ID NO: 20), KKKRKR (SEQ ID NO: 21), R5 (RRRRR) (SEQID NO: 22), R6 (RRRRRR) (SEQ ID NO: 23), and the like, or anycombination thereof, but not be limited thereto.

Some of the basic peptides have been known to have a nuclear membranepenetrating activity; however, none of them has been known to have acell membrane penetrating activity. In the present invention, the basicpeptide is fused with a hydrophobic peptide, to produce a cellpenetrating fusion peptide, thereby considerably increasing the cellmembrane penetrating effect of the hydrophobic peptide and thusincreases the cell penetrating ability of the fusion peptide comprisinggranzyme B.

The basic peptide may be linked (e.g., covalently) to the N-terminus orthe C-terminus of the hydrophobic peptide, or respectively linked toboth of the N-terminus and the C-terminus of the hydrophobic peptide(when two or more basic peptides are included). One particularembodiment, in order to more increase the cell membrane penetrability,the basic peptide may be linked to the N-terminus or the C-terminus, forexample the C-terminus, of the hydrophobic peptide. When two or morebasic peptides are respectively linked to both termini of thehydrophobic peptide, the basic peptides may be the same with ordifferent from one another.

In a particular embodiment, the cell penetrating fusion peptide maycomprise or consist essentially of:

a hydrophobic peptide and a basic peptide which is linked to theC-terminus of the hydrophobic peptide (that is, in the cell penetratingfusion peptide, a hydrophobic peptide is located at a N-terminal partand a basic peptide is located at a C-terminal part), or

a hydrophobic peptide and a basic peptide which is linked to N-terminusof the hydrophobic peptide (that is, in the cell penetrating fusionpeptide, a hydrophobic peptide is located at a C-terminal part and abasic peptide is located at a N-terminal part.

To increase the cell membrane penetrability of the cell penetratingfusion peptide, the cell penetrating fusion peptide may comprise ahydrophobic peptide and a basic peptide which is linked to theC-terminus of the hydrophobic peptide.

The cell penetrating fusion peptide may exhibit more excellent cellpenetrability than pre-existing cell penetrating peptides, and thus, itmay be useful in intracellular delivery of the fusion protein comprisinggranzyme B or a fragment thereof.

The cleavage site of a peptidase or protease may be located between thecell penetrating peptide and a targeting moiety in a fusion protein, andmay be capable of being cleaved under proper conditions (e.g., presenceand/or activation of the peptidase or protease). By such cleavage, acell penetrating peptide can be released from a targeting moiety havingrelatively large molecular weight, whereby the cell penetrating peptidecan exhibit a cell penetrating activity, thereby achieving a cellpenetration and intracellular delivery of granzyme B which is attached(linked) to the cell penetrating peptide.

In order to ensure the specificity of granzyme B to a cancer cell, thecleavage site may be cleaved by a peptidase or protease presentspecifically in a cancer cell. The peptidase or protease presentspecifically in a cancer cell may be a cancer cell-specific cleavageenzyme which is secreted specifically in a cancer cell or locatedspecifically on cell membrane of a cancer cell (e.g., exposed to outsideof cell membrane), and for example, endopeptidase. The cancercell-specific cleavage enzyme may be at least one selected from thegroup consisting of matrix metalloproteinase (MMP; e.g., MMP1, MMP2,MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16,MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26,MMP27, MMP28, etc.), cathepsin C, cathepsin H, urokinase-typeplasminogen activator (uPA), and the like, or any combination thereof.The cleavage site may be any cleavage (recognition) site of the cancercell-specific cleavage enzyme. In a particular example, the cleavagesite may be a cleavage (recognition) site of MMP9 (e.g., SGKIPRTLTA; SEQID NO: 35), but not be limited thereto.

The targeting moiety may be a domain capable of specifically targeting atarget cell or target tissue, and for example, it may be a substancecapable of targeting a specific cell such as a cancer cell or a specifictissue such as a cancer tissue. One or more targeting moieties may beincorporated into the fusion protein comprising granzyme B. For example,the targeting moiety may be comprised at both of C-terminus andN-terminus of a fusion protein comprising granzyme B, where thetargeting moieties at C-terminus and N-terminus may be the same with ordifferent to each other, but not be limited thereto. The targetingmoiety may be at least one selected from the group consisting of anantibody, an antigen-binding fragment of an antibody, a protein scaffoldsuch as a DARPin, and the like, or any combination thereof. The antibodyor the protein scaffold may be one specifically recognizing or bindingto at least one selected from the group consisting of signaltransduction substances (e.g., various growth factors, etc.), receptors(e.g., receptor tyrosine kinase proteins, etc.), and the like, which arepresent or overexpressed specifically in a target cell (e.g., a cancercell). The protein scaffold may be a protein construct characterized byhaving a similar structure to a protein or specifically recognizing orbinding a specific protein or a specific cell. For example, the proteinscaffold may be selected from the group consisting of a DARPin, anaffibody, a lasso scaffold, a cyclotide, a knottin, an avimer (short foravidity multimer), a Kunitz domain, an anticalin, an adnectin, apronectin, a fynomer, a nanofitin, an affilin, and the like, or anycombination thereof, but not limited thereto.

Examples of the growth factor may selected from the group consisting ofepidermal growth factor (EGF), platelet-derived growth factor (PDGF),fibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), and the like. Examples of the receptor tyrosine kinase proteinmay include receptors of various growth factors, and for example, beselected from the group consisting of an ErbB family such as epidermalgrowth factor receptor (EGFR), HER2, HER3, etc., insulin receptor,platelet-derived growth factor receptor (PDGFR), fibroblast growthfactor receptor (FGFR), vascular endothelial growth factor receptor(VEGFR), hepatocyte growth factor receptor (HGFR) such as c-Met,tropomyosin-receptor-kinase (Trk) receptor, Ephrin (Eph) receptor, AXLreceptor, Leukocyte receptor tyrosine kinase (LTK) receptor, TIEreceptor, receptor tyrosine kinase-like orphan (ROR) receptor, discoidindomain receptor (DDR), RET receptor, KLG receptor, related to receptortyrosine kinase (RYK) receptor, Muscle-Specific Kinase (MuSK) receptor,and the like.

The antibody may be from any subtype immunoglobulin (IgA, IgD, IgE, IgG(IgG1, IgG2, IgG3, IgG4), or IgM), which recognizes at least oneselected from the group consisting of various signal transductionsubstances, various receptors, and the like, present or overexpressedspecifically in a target cell, as an antigen. The antigen-bindingfragment may be a polypeptide comprising a part of an antibody, which isresponsible for specific binding to the antigen. The antigen-bindingfragment may be a heavy chain CDR (complementarity determining region),a light chain CDR, a heavy chain variable region, a light chain variableregion, or a combination thereof (e.g., scFv, (scFv)2, scFv-Fc, Fab,Fab′, or F(ab′)2). In a particular embodiment, the targeting moiety maybe an antigen-binding fragment of an antibody, such as scFv or scFv-Fc.

DARPin (designed ankyrin repeat protein) refers to an antibody mimeticprotein having high specificity and high binding affinity to a targetprotein, which is prepared via genetic engineering. DARPin is originatedfrom natural ankyrin protein, and has a structure comprising at least 2ankyrin repeat motifs, for example, comprising at least 3, 4 or 5ankyrin repeat motifs. The DARPin can have any suitable molecular weightdepending on the number of repeat motifs. For example, the DARPinsincluding 3, 4 or 5 ankyrin repeat motifs may have a molecular weight ofabout 10 kDa, about 14 kDa, or about 18 kDa, respectively. DARPinincludes a structural core that provides structure and a target bindingportion that resides outside of the core and binds to a target. Thestructural core includes a conserved amino acid sequence and the targetbinding portion includes an amino acid sequence that differs dependingon the target.

Since the DARPin has high affinity to an antigen (target), and higherstability and smaller molecular weight, it has advantageous properties(such as pharmacokinetic (PK) properties in the living body) andstability in the living body. In addition, the DARPin can be readilyfused with other proteins. Therefore, the DARPin can be useful inpreparing a fusion protein having excellent properties such as increasedstability in vivo

The fusion protein may comprise at least one DARPin, for example, about1 to about 10, about 1 to about 5, or about 1 to about 3 DARPins, whichinclude the same amino acid sequence, or at least two kinds of DARPins,for example, about 2 to about 10, about 2 to about 5, or about 2 toabout 3 kinds of DARPins, which include different amino acid sequencesand target the same or different antigens.

Examples of DARPins are summarized in the following table and thenucleotide sequences thereof are illustrated in FIGS. 5A to 5G and SEQID NOs: 37-68:

Target protein DARPins Human IgG1-Fc I_01/02/07/11/13/19 TNF-alphaT_01/02/07/08/09/16/25/27/37/40 ErbB1 (EGFR) E_01/67/68/69 ErbB2 (1-509)9_16/26/29 ErbB2 (1-631) H_14 ErbB4 B4_01/02/07/33/45/50/58 CitScp34_15/16

In an embodiment, the targeting moiety may be an anti-EGFR DARPin whichtargets EGFR. The anti-EGFR DARPin may be any DARPin having DARPin's ownunique structure and specifically binding to EGFR. For example, theanti-EGFR DARPin may be at least one selected from the group consistingof the following 4 anti-EGFR DARPins:

anti-EGFR DARPin-01 (SEQ ID NO: 30):dlgkklleaaragqddevrilmangadvnaddtwgwtplhlaayqghleivevllkngadvnaydyigwtplhlaadghleivevllkngadvnasdyigdtplhlaahnghleivevllkhgadvnaqdkfgktafd isidngnedlaeilqanti-EGFR DARPin-67 (SEQ ID NO: 31):dlgkklleaaragqddevrilmangadvnatdndgntplhlsawighleivevllkhgadvnaddllgmtplhlaadtghleivevllkygadvnardtrgktplhlaardghleivevllkhdadvnaqdkfgktaf disidngnedlaeilqanti-EGFR DARPin-68 (SEQ ID NO: 32):dlgkklleaaragqddevrilmangadvnafdywgmtplhlaadnghleivevllkhgadvnasdnfgftplhlaafyghleivevllkhgadvnafdmwgntplhlaaqnghleivevllkngadvnaqdkfgktaf disidngnedlaeilqanti-EGFR DARPin-69 (SEQ ID NO: 33):dlgkklleaaragqddevrilmangadvnaddnagrtplhlaanfghleivevllkngadvnakghhcntplhlaawaghleivevllkygadvnadddegytplhlaadigdleivevllkygadvnawdmygrtplhlaasaghleivevllkygadvnaqdkfgktafdisidngnedlae ilq

In the fusion protein, granzyme B may be located so that it is exposedat N-terminus of the fusion peptide, in order to be activated by anenzyme specifically present in a cancer cell, and the cleavage site of apeptidase or protease may be located between a cell penetrating peptideand targeting moiety, and cleaved when specifically reaching a cancercell by the targeting moiety, thereby releasing the cell penetratingpeptide from the targeting moiety to activate a cell membranepenetrating activity of the cell penetrating peptide. In addition,granzyme B may be linked to the cell penetrating peptide so that itpenetrates through a cell membrane together with the activated cellpenetrating peptide and moves inside of a cell. Therefore, the fusionprotein may comprise or consist essentially of, in order from N-terminusto C-terminus, (1) granzyme B, (2) a cell penetrating peptide (CPP), (3)a cleavage site of a peptidase or protease, and (4) a targeting moiety.As described above, when granzyme B is a fragment of granzyme Bcomprising an active region of granzyme B and a cleavage sequence, thefusion protein may comprise or consist essentially of, in order fromN-terminus to C-terminus, (1′) a fragment of ‘(a cleavage sequence)-(anactive region of granzyme B)’, (2) a cell penetrating peptide (CPP), (3)a cleavage site of a peptidase or protease, and (4) a targeting moiety.

The components of a fusion protein (i.e., granzyme B, a cell penetratingpeptide, a cleavage site of a peptidase or protease, and a targetingmoiety), or a hydrophobic peptide and a basic peptide of a cellpenetrating fusion peptide, may be independently linked to each otherthrough a peptide linker or directly linked to each other with nolinker. The peptide linker may be a peptide comprising about 1 to about20 amino acids or about 2 to about 10 amino acids. The peptide linkermay comprise at least one amino acid residue, and when the peptidelinker comprises two or more amino acid residues, each residue may beindependently selected from the group consisting of Gly, Asn, Ser, Thr,Ala, and the like. For example, the peptide linker may be (GS)n (whereinn is an integer from 1 to 5), but not be limited thereto. Severalappropriate amino acid sequences usefully employed as the peptide linkerare well known to the relevant art.

Each of the components may be linked to each other via a covalent bondsuch as a peptide bond, which is generally present in a protein.

granzyme B comprised in a fusion protein has a very strong cytotoxicity,allowing to effectively kill cancer cells, however it may exhibit afatal cytotoxicity to normal cells. Therefore, a targeting to a cancercell and a target-specific activity of granzyme B are very important inthe fusion protein. For this, the fusion protein employs a multiplesafety mechanism for a cancer cell specific activation of granzyme B,comprising (1) a targeting to a cancer cell by a targeting moiety, (2) acancer cell specific cleavage at a cleavage site and an activation of acell penetrating peptide by such cleavage, and (3) a use of granzyme B,which is activated by cathepsin present specifically in or by itself acancer cell. Therefore, the fusion protein can achieve an increasedefficacy of granzyme B in delivery to a cancer cell, cancer cellspecific cell membrane penetration, and/or intracellular delivery into acancer cell, and thus, it can be useful for intracellular delivery ofgranzyme B or for killing a cancer cell.

Another embodiment provides a pharmaceutical composition comprising afusion protein and a carrier.

Another embodiment provides a composition for cell membrane penetrationof granzyme B, comprising a fusion protein. Another embodiment providesa composition for intracellular delivery of granzyme B, comprising afusion protein. The cell to which the composition is delivered may be acell of a diseased region such as a cancer cell.

Another embodiment provides a method for intracellular delivery (or cellmembrane penetration) of granzyme B using a fusion protein. The methodfor intracellular delivery or cell membrane penetration may compriseadministering a fusion protein to a subject in need of intracellulardelivery or cell membrane penetration of granzyme B.

An embodiment provides an anticancer composition comprising a fusionprotein as an active ingredient. Another embodiment provides a method ofpreventing and/or treating a cancer comprising administering a fusionprotein to a subject in need of preventing and/or treating a cancer. Inthe method, the fusion protein may be administered in a pharmaceuticallyeffective amount. The method may further comprise a step of identifyinga subject in need of preventing and/or treating a cancer, prior to thestep of administering. The step of identifying may comprise determiningwhether or not a subject needs prevention and/or treatment of a cancer(e.g., a subject is suffered from a cancer).

The subject may be any animal selected from mammals such as primatesincluding human, monkeys, etc., rodents including rats, mice, etc., andthe like; a cell, a tissue, or body fluid (e.g., blood, serum) derived(isolated) from the animal; or a culture thereof. The subject may be ananimal, or a cell, a tissue, or body fluid derived (isolated) from theanimal, which is in need of delivery (e.g., intracellular delivery) ofgranzyme B included in the fusion protein.

The fusion protein may be administered to a subject in need ofadministration of granzyme B, via oral or parenteral route, oradministered by being contacted with a cell, tissue, or body fluidisolated from the subject.

The fusion protein or the pharmaceutically composition may furtherinclude a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be any one that is commonly used in formulationof nucleic acid containing drugs, and may be, but not limited to, atleast one selected from the group consisting of lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate,alginates, gelatin, calcium silicate, micro-crystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, mineral oil, and the like. The pharmaceutically compositionmay further include at least one selected from the group consisting of adiluent, an excipient, a lubricant, a wetting agent, a sweetener, aflavor enhancer, an emulsifying agent, a suspension agent, apreservative, and the like.

The fusion protein or the pharmaceutically composition may beadministered via oral or parenteral route. Parenteral administration maybe performed by intravenous injection, subcutaneous injection, muscularinjection, intraperitoneal injection, endothelial administration, localadministration, intranasal administration, intrapulmonaryadministration, and/or rectal administration. Since oral administrationleads to digestion of proteins or peptides, an active ingredient in thecompositions for oral administration must be coated or formulated toprevent digestion in stomach.

In addition, the fusion protein may be in a form of solution in oil oran aqueous medium, suspension, syrup, or emulsifying solution form, ormay be formulated into a form of an extract, powders, granules, a tabletor a capsule. The cell membrane penetrating conjugate may furtherinclude a dispersing agent and/or a stabilizing agent for itsformulation.

Another embodiment provides a method of improving (increasing) a cellmembrane penetrability of granzyme B, wherein the method comprisespreparing a fusion protein comprising (1) granzyme B, (2) a cellpenetrating peptide (CPP), (3) a cleavage site, and (4) a targetingmoiety. Another embodiment provides a method of preparing granzyme Bderivative having increased cell membrane penetrability, wherein themethod comprises preparing s fusion protein comprising (1) granzyme B,(2) a cell penetrating peptide (CPP), (3) a cleavage site, and (4) atargeting moiety. The step of preparing a fusion protein may beconducted ex vivo. Each component of the fusion protein may beindependently linked to each other through a peptide linker or directlylinked to each other with no linker, as described above.

The preparation of the fusion protein may be performed by any generalmethod for preparing a protein, such as a chemical synthesis of aprotein, a protein expression using an expression (recombinant) vectorcomprising (carrying) polynucleotides encoding each component of thefusion protein (in a proper host cell), and the like. Thus, alsoprovided herein is a polynucleotide encoding the fusion proteindescribed herein, optionally in an expression (recombinant) vector, aswell as a method of producing the fusion protein comprising expressingthe polynucleotide encoding the fusion protein in a cell or culturing arecombinant cell comprising (harboring) the polynucleotide encoding thefusion protein to express the polynucleotide.

Each component of the fusion protein, i.e., granzyme B, a cellpenetrating peptide, a cleavage site, and a targeting moiety, isdescribed above.

As used herein, the term “vector” refers to a means for expressing agene of interest in a host cell, as exemplified by a plasmid vector, acosmid vector, and a viral vector, such as a bacteriophage vector, anadenovirus vector, a retrovirus vector and an adeno-associated virusvector. The recombinant vector may be constructed from well-knownplasmids (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290,pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ6l, pLAFR1, pHV14, pGEX series,pET series, pUC19, etc.), phages (for example, λgt4λB, λ-Charon, λΔz1,M13, etc.) or viruses (for example, SV40, etc.) by known manipulation(genetic engineering) techniques.

In the recombinant vector, the polynucleotide encoding the proteinconjugate may be operatively linked to a promoter. The term “operativelylinked” is intended to pertain to a functional linkage between anucleotide sequence of interest and an expression regulatory element(for example, a promoter sequence) so that the expression of thenucleotide sequence of interest is governed by the regulatory element.For instance, when it is “operatively linked” to the regulatory element,the nucleotide sequence of interest can be transcribed and/or translatedunder the control of the regulatory element.

The recombinant vector may be constructed typically as a cloning vectoror an expression vector. For recombinant expression vectors, a vectortypically available for expressing a foreign protein in plant, animal ormicroorganism cells may be employed. Various methods well known in theart may be used for the construction of recombinant vectors.

For use in hosts, such as prokaryotic or eukaryotic cells, therecombinant vector may be constructed appropriately. For example, when avector is constructed as an expression vector for use in a prokaryotichost, the vector typically includes a strong promoter for transcription(e.g., a pLλ promoter, a CMV promoter, a trp promoter, a lac promoter, atac promoter, a T7 promoter, etc.), a ribosomal binding side forinitiating translation, and transcriptional/translational terminationsites. On the other hand, an expression vector for use in a eukaryotichost includes an origin of replication operable in a eukaryotic cell,such as, but not limited to, an f1 origin of replication, an SV40 originof replication, a pMB1 origin of replication, an adeno origin ofreplication, an AAV origin of replication, a BBV origin of replication.In addition, the expression vector typically includes a promoter derivedfrom mammalian cells (for example, metallothionein promoter) or frommammalian viruses (for example, adenovirus late promoter, vaccinia virus7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoterof HSV), and a polyadenylation sequence as a transcription terminationsequence.

The recombinant cell may be prepared by introducing the recombinantvector into a suitable host cell. So long as it allows for thesequential cloning and expression of the recombinant vector in a stablemanner, any host cell known in the art may be employed. Examples of theprokaryotic host cell available may be at least one selected from thegroup consisting of E. coli JM109, E. coli BL21, E. coli RR1, E. coliLE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus spp. such asBacillus subtilis and Bacillus thuringiensis, and enterobacteriaceaestrains such as Salmonella typhimurium, Serratia marcescens, and variousPseudomonas species. Eukaryotic host cells to be transformed may be atleast one selected from the group consisting of Saccharomycescerevisiae, insect cells, plant cells and animal cells including Sp2/0,CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293,HepG2, Huh7, 3T3, RIN, and MDCK, but not be limited thereto.

Using a method well known in the art, the polynucleotide or arecombinant vector carrying the polynucleotide may be introduced(incorporated) into a host cell. This transformation is carried outthrough CaCl₂ or electroporation when the host cell is prokaryotic. Foreukaryotic host cells, the genetic introduction may be achieved using,but not limited to, microinjection, calcium phosphate precipitation,electroporation, liposome-mediated transfection, or particlebombardment.

To select a (recombinant vector) transformed host cell, advantage may betaken of the phenotype attributed to a selection marker according to amethod known in the art. For example, when the selection marker is agene resistant to a certain antibiotic, the host cells may be grown inthe presence of the antibiotic in a medium to select a transformant ofinterest.

The fusion protein capable of an effective intracellular delivery ofgranzyme B specific to a cancer cell, and the pharmaceutical composition(anticancer composition) comprising the fusion protein may exhibit a“first-in class” anticancer activity, which is distinguished frompre-existing anticancer agents, and thus, they are expected to be usefulas an effective anticancer drug. For example, pharmaceutical compositionmay have a large effect on almost all types of solid tumors such as lungcancer, breast cancer, etc., and be also expected to have a good effecton metastasis.

EXAMPLES

Hereafter, the present invention will be described in detail byexamples.

The following examples are intended merely to illustrate the inventionand are not construed to restrict the invention.

Example 1 Preparation of a Fusion Protein

1.1. Preparation of GZB-MTS-BAA-M9R-D(E) A fusion protein (amino acidsequence: SEQ ID NO: 1; nucleotide sequence: SEQ ID NO: 2), whichcomprises, in order from N-terminus to C-terminus, “granzyme B(GZB)-cell penetrating fusion peptide [membrane-translocation sequence(MTS)-basic amino acid (BAA)]-cleavage site (M9R)-targeting moiety [EGFRDARPin (D(E))], was prepared (the first construct in FIG. 1). The aminoacid sequence of KKKRK (SEQ ID NO: 15), which is a representativenuclear transfer signal, was used as “BAA”.

The nucleotide sequence of SEQ ID NO: 2 was transfected into E. coliBL21 (DE3) cell and expressed, to produce the fusion protein having theamino acid sequence of SEQ ID NO: 1. In detail, the polynucleotides werecloned to pET21b vector (Novagen) using the restriction enzymes NdeI(NEB) and XhoI (NEB) to construct recombinant vectors pET21b. Theserecombinant vectors were introduced into an E. coli strain (BL21 (DE3)Codon Plus-RIPL; Invitrogen).

The transfected cells were cultured in LB media. When the O.D. value ofabsorbance at 600 nm reaches 0.5, 1 mM of IPTG(isopropyl-β-D-thio-galactoside) was added to the cell culture, whichwas further cultured at 18° C. for 16 hours. The obtained cultured cellswere crushed by sonication in the presence of 20 mM Tris-HCl buffersolution (pH 7.4) supplemented with 10% glycerol and 0.25M NaCl, andcentrifuged at 10,000 g, to obtain a supernatant. The obtainedsupernatant was applied to Ni²⁺-NTA superflow column (Qiagen) which isequilibrated with the buffer solution. The column was washed with awashing buffer (20 mM Tris-HCl, pH 7.4, 10% glycerol. and 1 M NaCl) inthe amount of 5-fold of the column volume, and then treated with aeluting buffer (20 mM Tris-HCl, pH 7.4, 10% glycerol, 0.25 M NaCl and0.2M imidazole), to elute the fusion protein. The fractions includingthe fusion protein were collected and salts comprised in the fractionswere removed using Amicon Ultra-15 Centrifugal Filter (Milipore), toconcentrate and purify the protein. The concentration of the purifiedprotein was measured using BSA as a standard substance.

The expression of the fusion protein was observed through SDS-PAGEassay, and the obtained results are shown in FIG. 2.

The prepared fusion protein (SEQ ID NO: 1) and its coding nucleotidesequence (SEQ ID NO: 2) are summarized in Table 1:

TABLE 1 Nucleotide Sequence N → C Amino Acid Sequenceencoding the Amino Acid Sequence His6 MRGSHHHHHHDYDIPTTATGCGCGGCAGCCATCACCATCACCATCACGATTACG ATATCCCAACGACC Dar(E)DLGKKLLEAARAGQDDEVRI GATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCGLMANGADVNADDTWGWTPL GGCCAGGATGATGAAGTGCGCATTCTGATGGCGAATHLAAYQGHLEIVEVLLKNGA GGTGCGGATGTTAACGCGGACGATACCTGGGGCTGGDVNAYDYIGWTPLHLAADGH ACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGGLEIVEVLLKNGADVNASDYIG AAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATGDTPLHLAAHNGHLEIVEVLLK TGAACGCGTATGATTATATTGGCTGGACCCCGCTGCAHGADVNAQDKFGKTAFDISID TCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG Linker GSGGCAGC TEV ENLYFQGS GAAAACCTGTATTTTCAGGGATCC Linker GSGS GGCAGCGGCAGCGZB MQPILLLLAFLLLPRADAGEII ATGCAGCCGATCCTGCTCCTCCTGGCGTTCCTGCTGCGGHEAKPHSRPYMAYLMIWD TGCCACGTGCTGACGCTGGTGAAATCATCGGTGGTCAQKSLKRCGGFLIQDDFVLTAA CGAAGCTAAACCGCACTCTCGTCCGTACATGGCTTACHCWGSSINVTLGAHNIKEQEP CTGATGATCTGGGACCAGAAATCTCTGAAACGTTGCGTQQFIPVKRPIPHPAYNPKNFS GTGGTTTCCTGATCCAGGACGACTTCGTTCTGACCGCNDIMLLQLERKAKRTRAVQP TGCTCACTGCTGGGGTTCTTCTATCAACGTTACCCTGLRLPSNKAQVKPGQTCSVAG GGTGCTCACAACATCAAAGAACAGGAACCGACCCAGWGQTAPLGKHSHTLQEVKMT CAGTTCATCCCGGTTAAACGTCCGATCCCGCACCCGGVQEDRKCESDLRHYYDSTIEL CTTACAACCCGAAAAACTTCTCTAACGACATCATGCTCVGDPEIKKTSFKGDSGGPLV GCTGCAGCTGGAACGTAAAGCTAAACGTACCCGTGCCNKVAQGIVSYGRNNGMPPR TGTTCAGCCGCTGCGTCTGCCGTCTAACAAAGCTCAGACTKVSSFVHWIKKTMKRH GTTAAACCGGGTCAGACCTGCTCTGTTGCTGGTTGGGGTCAGACCGCTCCGCTGGGTAAACACTCTCACACCCTGCAGGAAGTTAAAATGACCGTTCAGGAAGACCGTAAATGCGAATCTGACCTGCGTCACTACTACGACTCTACCATCGAACTGTGCGTTGGTGACCCGGAAATCAAAAAAACCTCTTTCAAAGGTGACTCTGGTGGTCCGCTGGTTTGCAACAAAGTTGCTCAGGGTATCGTTTCTTACGGTCGTAACAACGGTATGCCGCCGCGTGCTTGCACCAAAGTTTCTTCTTTCGTTCACTGGATCAAAAAAACCATGAAAC GTCAC Linker GS GGCAGC MTSAAVALLPAVLLALLAP GCCGCGGTAGCGCTGCTCCCGGCGGTCCTGCTGGCCT TGCTGGCGCCC BAAKKKRK AAAAAGAAGCGCAAG linker GS GGCAGC N9R SGKIPRTLTAAGCGGCAAAATTCCGCGTACCCTGACCGCG linker AS GCTAGC Dar(E)DLGKKLLEAARAGQDDEVRI GATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCGLMANGADVNADDTWGWTPL GGCCAGGATGATGAAGTGCGCATTCTGATGGCGAATHLAAYQGHLEIVEVLLKNGA GGTGCGGATGTTAACGCGGACGATACCTGGGGCTGGDVNAYDYIGWTPLHLAADGH ACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGGLEIVEVLLKNGADVNASDYIG AAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATGDTPLHLAAHNGHLEIVEVLLK TGAACGCGTATGATTATATTGGCTGGACCCCGCTGCAHGADVNAQDKFGKTAFDISID TCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG (wherein,the part “His6-D(E)-TEV” at N-terminus was inserted merely forexpression and identification, and has no effect on the activity of thefusion protein, and thus, the part was removed in the fusion proteinused in the activity assay)

1.2. Preparation of GEGZB-MTS-BAA-M9R-Dar(EGFR)-MTS-BAA

A fusion protein (amino acid sequence: SEQ ID NO: 3; nucleotidesequence: SEQ ID NO: 4), which comprises, in order from N-terminus toC-terminus, “granzyme B fragment (GEGZB)-cell penetrating fusion peptide[MTS-BAA]-cleavage site (M9R)-targeting moiety [EGFR DARPin(Dar(EGFR))]-cell penetrating fusion peptide [MTS-BAA]”, was prepared(the second construct in FIG. 1). The amino acid sequence of KKKRK (SEQID NO: 15), which is a representative nuclear transfer signal, was usedas “BAA”.

The preparation of the fusion protein was performed referring to Example1.1. The prepared fusion protein (SEQ ID NO: 3) and its codingnucleotide sequence (SEQ ID NO: 4) are summarized in Table 2:

TABLE 2 Nucleotide Sequence  N → C Amino Acid Sequenceencoding the Amino Acid Sequence His6 MRGSHHHHHHDYDIPTTATGCGCGGCAGCCATCACCATCACCATCACGATTACG ATATCCCAACGACC DarDLGKKLLEAARAGQDDEVRI GATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR)LMANGADVNADDTWGWTPL GGCCAGGATGATGAAGTGCGCATTCTGATGGCGAATHLAAYQGHLEIVEVLLKNGA GGTGCGGATGTTAACGCGGACGATACCTGGGGCTGGDVNAYDYIGWTPLHLAADGH ACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGGLEIVEVLLKNGADVNASDYIG AAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATGDTPLHLAAHNGHLEIVEVLLK TGAACGCGTATGATTATATTGGCTGGACCCCGCTGCAHGADVNAQDKFGKTAFDISID TCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG linker GSGGCAGC TEV- ENLYFQGEIIGGHEAKPHSRP GAAAACCTGTATTTTCAGGGAGAAATCATCGGTGGTCGEGZB YMAYLMIWDQKSLKRCGGFL ACGAAGCTAAACCGCACTCTCGTCCGTACATGGCTTAIQDDFVLTAAHCWGSSINVTL CCTGATGATCTGGGACCAGAAATCTCTGAAACGTTGCGAHNIKEQEPTQQFIPVKRPIP GGTGGTTTCCTGATCCAGGACGACTTCGTTCTGACCGHPAYNPKNFSNDIMLLQLERK CTGCTCACTGCTGGGGTTCTTCTATCAACGTTACCCTGAKRTRAVQPLRLPSNKAQVK GGTGCTCACAACATCAAAGAACAGGAACCGACCCAGPGQTCSVAGWGQTAPLGKHS CAGTTCATCCCGGTTAAACGTCCGATCCCGCACCCGGHTLQEVKMTVQEDRKCESDL CTTACAACCCGAAAAACTTCTCTAACGACATCATGCTRHYYDSTIELCVGDPEIKKTSF GCTGCAGCTGGAACGTAAAGCTAAACGTACCCGTGCKGDSGGPLVCNKVAQGIVSY TGTTCAGCCGCTGCGTCTGCCGTCTAACAAAGCTCAGGRNNGMPPRACTKVSSFVHW GTTAAACCGGGTCAGACCTGCTCTGTTGCTGGTTGGG IKKTMKRHGTCAGACCGCTCCGCTGGGTAAACACTCTCACACCCTGCAGGAAGTTAAAATGACCGTTCAGGAAGACCGTAAATGCGAATCTGACCTGCGTCACTACTACGACTCTACCATCGAACTGTGCGTTGGTGACCCGGAAATCAAAAAAACCTCTTTCAAAGGTGACTCTGGTGGTCCGCTGGTTTGCAACAAAGTTGCTCAGGGTATCGTTTCTTACGGTCGTAACAACGGTATGCCGCCGCGTGCTTGCACCAAAGTTTCTTCTTTCGTTCACTGGATCAAAAAAACCATGAAAC GTCAC linker GS GGCAGC MTSAAVALLPAVLLALLAP GCCGCGGTAGCGCTGCTCCCGGCGGTCCTGCTGGCCT TGCTGGCGCCC BAAKKKRK AAAAAGAAGCGCAAG linker GS GGCAGC N9R SGKIPRTLTAAGCGGCAAAATTCCGCGTACCCTGACCGCG NheI AS GCTAGC Dar DLGKKLLEAARAGQDDEVRIGATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR) LMANGADVNADDTWGWTPLGGCCAGGATGATGAAGTGCGCATTCTGATGGCGAAT HLAAYQGHLEIVEVLLKNGAGGTGCGGATGTTAACGCGGACGATACCTGGGGCTGG DVNAYDYIGWTPLHLAADGHACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGG LEIVEVLLKNGADVNASDYIGAAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATG DTPLHLAAHNGHLEIVEVLLKTGAACGCGTATGATTATATTGGCTGGACCCCGCTGCA HGADVNAQDKFGKTAFDISIDTCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG linker GSGGCAGC TAT YGRKKRRQRRR TATGGCCGCAAGAAACGTCGCCAGCGCCGTCGT NLS KKKRKAAGAAAAAACGTAAG (wherein, the part “His6-D(E)-TEV” at N-terminus wasinserted merely for expression and identification, and has no effect onthe activity of the fusion protein, and thus, the part was removed inthe fusion protein used in the activity assay)

1.3. Preparation of GEGZB-MTS-BAA-M9R-Dar(EGFR)

A fusion protein (amino acid sequence: SEQ ID NO: 5; nucleotidesequence: SEQ ID NO: 6), which comprises, in order from N-terminus toC-terminus, “granzyme B fragment (GEGZB)-cell penetrating fusion peptide[MTS-BAA]-cleavage site (M9R)-targeting moiety [EGFR DARPin(Dar(EGFR))]”, was prepared (the third construct in FIG. 1). The aminoacid sequence of KKKRK (SEQ ID NO: 15), which is a representativenuclear transfer signal, was used as “BAA”.

The preparation of the fusion protein was performed referring to Example1.1. The prepared fusion protein (SEQ ID NO: 5) and its codingnucleotide sequence (SEQ ID NO: 6) are summarized in Table 3:

TABLE 3 Nucleotide Sequence N → C Amino Acid Sequenceencoding the Amino Acid Sequence His6 MRGSHHHHHHDYDIPTTATGCGCGGCAGCCATCACCATCACCATCACGATTACG ATATCCCAACGACC Dar DLGKKLLEAARAGQDDEVRI GATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR)LMANGADVNADDTWGWTPL GGCCAGGATGATGAAGTGCGCATTCTGATGGCGAATHLAAYQGHLEIVEVLLKNGA GGTGCGGATGTTAACGCGGACGATACCTGGGGCTGGDVNAYDYIGWTPLHLAADGH ACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGGLEIVEVLLKNGADVNASDYIG AAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATGDTPLHLAAHNGHLEIVEVLLK TGAACGCGTATGATTATATTGGCTGGACCCCGCTGCAHGADVNAQDKFGKTAFDISID TCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG linker GSGGCAGC TEV- ENLYFQGEIIGGHEAKPHSRP GAAAACCTGTATTTTCAGGGAGAAATCATCGGTGGTCGEGZB YMAYLMIWDQKSLKRCGGFL ACGAAGCTAAACCGCACTCTCGTCCGTACATGGCTTAIQDDFVLTAAHCWGSSINVTL CCTGATGATCTGGGACCAGAAATCTCTGAAACGTTGCGAHNIKEQEPTQQFIPVKRPIP GGTGGTTTCCTGATCCAGGACGACTTCGTTCTGACCGHPAYNPKNFSNDIMLLQLERK CTGCTCACTGCTGGGGTTCTTCTATCAACGTTACCCTGAKRTRAVQPLRLPSNKAQVK GGTGCTCACAACATCAAAGAACAGGAACCGACCCAGPGQTCSVAGWGQTAPLGKHS CAGTTCATCCCGGTTAAACGTCCGATCCCGCACCCGGHTLQEVKMTVQEDRKCESDL CTTACAACCCGAAAAACTTCTCTAACGACATCATGCTRHYYDSTIELCVGDPEIKKTSF GCTGCAGCTGGAACGTAAAGCTAAACGTACCCGTGCKGDSGGPLVCNKVAQGIVSY TGTTCAGCCGCTGCGTCTGCCGTCTAACAAAGCTCAGGRNNGMPPRACTKVSSFVHW GTTAAACCGGGTCAGACCTGCTCTGTTGCTGGTTGGG IKKTMKRHGTCAGACCGCTCCGCTGGGTAAACACTCTCACACCCTGCAGGAAGTTAAAATGACCGTTCAGGAAGACCGTAAATGCGAATCTGACCTGCGTCACTACTACGACTCTACCATCGAACTGTGCGTTGGTGACCCGGAAATCAAAAAAACCTCTTTCAAAGGTGACTCTGGTGGTCCGCTGGTTTGCAACAAAGTTGCTCAGGGTATCGTTTCTTACGGTCGTAACAACGGTATGCCGCCGCGTGCTTGCACCAAAGTTTCTTCTTTCGTTCACTGGATCAAAAAAACCATGAAAC GTCAC linker GS GGCAGC MTSAAVALLPAVLLALLAP GCCGCGGTAGCGCTGCTCCCGGCGGTCCTGCTGGCCT TGCTGGCGCCC BAAKKKRK AAAAAGAAGCGCAAG linker GS GGCAGC N9R SGKIPRTLTAAGCGGCAAAATTCCGCGTACCCTGACCGCG NheI AS GCTAGC Dar DLGKKLLEAARAGQDDEVRIGATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR) LMANGADVNADDTWGWTPLGGCCAGGATGATGAAGTGCGCATTCTGATGGCGAAT HLAAYQGHLEIVEVLLKNGAGGTGCGGATGTTAACGCGGACGATACCTGGGGCTGG DVNAYDYIGWTPLHLAADGHACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGG LEIVEVLLKNGADVNASDYIGAAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATG DTPLHLAAHNGHLEIVEVLLKTGAACGCGTATGATTATATTGGCTGGACCCCGCTGCA HGADVNAQDKFGKTAFDISIDTCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG (wherein,the part “His6-D(E)-TEV” at N-terminus was inserted merely forexpression and identification, and has no effect on the activity of thefusion protein, and thus, the part was removed in the fusion proteinused in the activity assay)

1.4. Preparation of IEPDGZB-MTS-BAA-M9R-Dar(EGFR)-MTS-BAA

A fusion protein (amino acid sequence: SEQ ID NO: 7; nucleotidesequence: SEQ ID NO: 8), which comprises, in order from N-terminus toC-terminus, “granzyme B fragment (IEPDGZB)-cell penetrating fusionpeptide [(MTS)-BAA]-cleavage site (M9R)-targeting moiety [EGFR DARPin(Dar(EGFR))]-cell penetrating fusion peptide [MTS-BAA]”, was prepared(the fourth construct in FIG. 1). The amino acid sequence of KKKRK (SEQID NO: 15), which is a representative nuclear transfer signal, was usedas “BAA”.

The preparation of the fusion protein was performed referring to Example1.1. The prepared fusion protein (SEQ ID NO: 7) and its codingnucleotide sequence (SEQ ID NO: 8) are summarized in Table 4:

TABLE 4 Nucleotide Sequence N → C Amino Acid Sequenceencoding the Amino Acid Sequence His6 MRGSHHHHHHDYDIPTTATGCGCGGCAGCCATCACCATCACCATCACGATTACG ATATCCCAACGACC DarDLGKKLLEAARAGQDDEVRI GATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR)LMANGADVNADDTWGWTPL GGCCAGGATGATGAAGTGCGCATTCTGATGGCGAATHLAAYQGHLEIVEVLLKNGA GGTGCGGATGTTAACGCGGACGATACCTGGGGCTGGDVNAYDYIGWTPLHLAADGH ACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGGLEIVEVLLKNGADVNASDYIG AAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATGDTPLHLAAHNGHLEIVEVLLK TGAACGCGTATGATTATATTGGCTGGACCCCGCTGCAHGADVNAQDKFGKTAFDISID TCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG linker GSGGCAGC TEV ENLYFQGS GAAAACCTGTATTTTCAGGGATCC linker GSGS GGCAGCGGCAGCIEPD- MGSIEPDIIGGHEAKPHSRPY ATGGGCAGCATCGAACCAGATATCATCGGTGGTCAC GZBMAYLMIWDQKSLKRCGGFLI GAAGCTAAACCGCACTCTCGTCCGTACATGGCTTACCQDDFVLTAAHCWGSSINVTL TGATGATCTGGGACCAGAAATCTCTGAAACGTTGCGGGAHNIKEQEPTQQFIPVKRPIP TGGTTTCCTGATCCAGGACGACTTCGTTCTGACCGCTHPAYNPKNFSNDIMLLQLERK GCTCACTGCTGGGGTTCTTCTATCAACGTTACCCTGGAKRTRAVQPLRLPSNKAQVK GTGCTCACAACATCAAAGAACAGGAACCGACCCAGCPGQTCSVAGWGQTAPLGKHS AGTTCATCCCGGTTAAACGTCCGATCCCGCACCCGGCHTLQEVKMTVQEDRKCESDL TTACAACCCGAAAAACTTCTCTAACGACATCATGCTGRHYYDSTIELCVGDPEIKKTSF CTGCAGCTGGAACGTAAAGCTAAACGTACCCGTGCTKGDSGGPLVCNKVAQGIVSY GTTCAGCCGCTGCGTCTGCCGTCTAACAAAGCTCAGGGRNNGMPPRACTKVSSFVHW TTAAACCGGGTCAGACCTGCTCTGTTGCTGGTTGGGG IKKTMKRHTCAGACCGCTCCGCTGGGTAAACACTCTCACACCCTGCAGGAAGTTAAAATGACCGTTCAGGAAGACCGTAAATGCGAATCTGACCTGCGTCACTACTACGACTCTACCATCGAACTGTGCGTTGGTGACCCGGAAATCAAAAAAACCTCTTTCAAAGGTGACTCTGGTGGTCCGCTGGTTTGCAACAAAGTTGCTCAGGGTATCGTTTCTTACGGTCGTAACAACGGTATGCCGCCGCGTGCTTGCACCAAAGTTTCTTCTTTCGTTCACTGGATCAAAAAAACCATGAAACG TCAC linker GS GGCAGC MTSAAVALLPAVLLALLAP GCCGCGGTAGCGCTGCTCCCGGCGGTCCTGCTGGCCT TGCTGGCGCCC BAAKKKRK AAAAAGAAGCGCAAG linker GS GGCAGC N9R SGKIPRTLTAAGCGGCAAAATTCCGCGTACCCTGACCGCG NheI AS GCTAGC Dar DLGKKLLEAARAGQDDEVRIGATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR) LMANGADVNADDTWGWTPLGGCCAGGATGATGAAGTGCGCATTCTGATGGCGAAT HLAAYQGHLEIVEVLLKNGAGGTGCGGATGTTAACGCGGACGATACCTGGGGCTGG DVNAYDYIGWTPLHLAADGHACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGG LEIVEVLLKNGADVNASDYIGAAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATG DTPLHLAAHNGHLEIVEVLLKTGAACGCGTATGATTATATTGGCTGGACCCCGCTGCA HGADVNAQDKFGKTAFDISIDTCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG linker GSGGCAGC TAT YGRKKRRQRRR TATGGCCGCAAGAAACGTCGCCAGCGCCGTCGT NLS KKKRKAAGAAAAAACGTAAG (wherein, the part “His6-D(E)-TEV” at N-terminus wasinserted merely for expression and identification, and has no effect onthe activity of the fusion protein, and thus, the part was removed inthe fusion protein used in the activity assay; and the sequence “IEPD”included at the N-terminus of the granzyme B fragment (IEPD-GZB) is anauto-cleavage sequence which induces auto-activation of granzyme B andcleavage by granzyme B to be removed, making the sequence “IIG” to beexposed on the N-terminus of the granzyme B fragment (IEPD-GZB)

1.5. Preparation of IEPDGZB-MTS-BAA-M9R-Dar(EGFR)

A fusion protein (amino acid sequence: SEQ ID NO: 9; nucleotidesequence: SEQ ID NO: 10), which comprises, in order from N-terminus toC-terminus, “granzyme B fragment (IEPDGZB)-cell penetrating fusionpeptide [MTS-BAA]-cleavage site (M9R)-targeting moiety [EGFR DARPin(Dar(EGFR))]”, was prepared (the fifth construct in FIG. 1). The aminoacid sequence of KKKRK (SEQ ID NO: 15), which is a representativenuclear transfer signal, was used as “BAA”.

The preparation of the fusion protein was performed referring to Example1.1. The prepared fusion protein (SEQ ID NO: 9) and its codingnucleotide sequence (SEQ ID NO: 10) are summarized in Table 5:

TABLE 5 Nucleotide Sequence N → C Amino Acid Sequenceencoding the Amino Acid Sequence His6 MRGSHHHHHHDYDIPTTATGCGCGGCAGCCATCACCATCACCATCACGATTACG ATATCCCAACGACC DarDLGKKLLEAARAGQDDEVRI GATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR)LMANGADVNADDTWGWTPL GGCCAGGATGATGAAGTGCGCATTCTGATGGCGAATHLAAYQGHLEIVEVLLKNGA GGTGCGGATGTTAACGCGGACGATACCTGGGGCTGGDVNAYDYIGWTPLHLAADGH ACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGGLEIVEVLLKNGADVNASDYIG AAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATGDTPLHLAAHNGHLEIVEVLLK TGAACGCGTATGATTATATTGGCTGGACCCCGCTGCAHGADVNAQDKFGKTAFDISID TCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG linker GSGGCAGC TEV ENLYFQGS GAAAACCTGTATTTTCAGGGATCC linker GSGS GGCAGCGGCAGCIEPD- MGSIEPDIIGGHEAKPHSRPY ATGGGCAGCATCGAACCAGATATCATCGGTGGTCAC GZBMAYLMIWDQKSLKRCGGFLI GAAGCTAAACCGCACTCTCGTCCGTACATGGCTTACCQDDFVLTAAHCWGSSINVTL TGATGATCTGGGACCAGAAATCTCTGAAACGTTGCGGGAHNIKEQEPTQQFIPVKRPIP TGGTTTCCTGATCCAGGACGACTTCGTTCTGACCGCTHPAYNPKNFSNDIMLLQLERK GCTCACTGCTGGGGTTCTTCTATCAACGTTACCCTGGAKRTRAVQPLRLPSNKAQVK GTGCTCACAACATCAAAGAACAGGAACCGACCCAGCPGQTCSVAGWGQTAPLGKHS AGTTCATCCCGGTTAAACGTCCGATCCCGCACCCGGCHTLQEVKMTVQEDRKCESDL TTACAACCCGAAAAACTTCTCTAACGACATCATGCTGRHYYDSTIELCVGDPEIKKTSF CTGCAGCTGGAACGTAAAGCTAAACGTACCCGTGCTKGDSGGPLVCNKVAQGIVSY GTTCAGCCGCTGCGTCTGCCGTCTAACAAAGCTCAGGGRNNGMPPRACTKVSSFVHW TTAAACCGGGTCAGACCTGCTCTGTTGCTGGTTGGGG IKKTMKRHTCAGACCGCTCCGCTGGGTAAACACTCTCACACCCTGCAGGAAGTTAAAATGACCGTTCAGGAAGACCGTAAATGCGAATCTGACCTGCGTCACTACTACGACTCTACCATCGAACTGTGCGTTGGTGACCCGGAAATCAAAAAAACCTCTTTCAAAGGTGACTCTGGTGGTCCGCTGGTTTGCAACAAAGTTGCTCAGGGTATCGTTTCTTACGGTCGTAACAACGGTATGCCGCCGCGTGCTTGCACCAAAGTTTCTTCTTTCGTTCACTGGATCAAAAAAACCATGAAACG TCAC linker GS GGCAGC MTSAAVALLPAVLLALLAP GCCGCGGTAGCGCTGCTCCCGGCGGTCCTGCTGGCCT TGCTGGCGCCC BAAKKKRK AAAAAGAAGCGCAAG linker GS GGCAGC N9R SGKIPRTLTAAGCGGCAAAATTCCGCGTACCCTGACCGCG NheI AS GCTAGC Dar DLGKKLLEAARAGQDDEVRIGATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCG (EGFR) LMANGADVNADDTWGWTPLGGCCAGGATGATGAAGTGCGCATTCTGATGGCGAAT HLAAYQGHLEIVEVLLKNGAGGTGCGGATGTTAACGCGGACGATACCTGGGGCTGG DVNAYDYIGWTPLHLAADGHACCCCACTGCATCTGGCCGCGTATCAGGGTCACCTGG LEIVEVLLKNGADVNASDYIGAAATCGTGGAGGTGCTGCTGAAAAACGGCGCGGATG DTPLHLAAHNGHLEIVEVLLKTGAACGCGTATGATTATATTGGCTGGACCCCGCTGCA HGADVNAQDKFGKTAFDISIDTCTGGCGGCGGATGGCCATCTGGAAATTGTGGAAGT NGNEDLAEILQGCTGCTGAAAAACGGCGCTGATGTTAATGCTAGCGATTATATTGGCGATACGCCGCTGCACCTGGCAGCGCATAACGGCCATCTGGAGATTGTTGAAGTTCTGCTGAAGCATGGCGCCGATGTGAATGCGCAGGATAAATTTGGCAAAACCGCGTTTGATATTAGCATTGATAACGGCAACGAA GATCTGGCGGAAATTCTGCAG (wherein,the part “His6-D(E)-TEV” at N-terminus was inserted merely forexpression and identification, and has no effect on the activity of thefusion protein, and thus, the part was removed in the fusion proteinused in the activity assay; and the sequence “IEPD” included at theN-terminus of the granzyme B fragment (IEPD-GZB) is an auto-cleavagesequence which induces auto-activation of granzyme B and cleavage bygranzyme B to be removed, making the sequence “IIG” to be exposed on theN-terminus of the granzyme B fragment (IEPD-GZB)

Example 2 Cancer Cell Proliferation Inhibition by Granzyme B ContainingFusion Protein

Anticancer effects of the prepared granzyme B containing fusion proteinswere examined in human colon cancer cell line, HCT116 (ATCC) and RKO(ATCC).

The cells were inoculated in RPMI medium (Gibco) supplemented with10%(v/v) FBS in each 96-well plate in the amount of 1×10³ cells per eachwell. In the next day, the cells were treated with each of granzyme Bcontaining fusion proteins ‘IEPDGZB-MTS-BAA-M9R-Dar(EGFR)-MTS-BAA’ (SEQID NO: 7), and ‘IEPDGZB-MTS-BAA-M9R-Dar(EGFR)’ (SEQ ID NO: 9), whereinthe concentration of each fusion protein was 0, 0.1, 0.2, 0.4, 0.8, or1.6 μM, and the amount of each fusion protein treated was 100 μL per awell. The cells were incubated in CO₂ incubator for 24 hours under thecondition of 37° C., and CO₂ 5%. After adding 80 μL of CellTiter-Gloreagent (Promega) to each well, luminescence was measured using EnVisionMultilabel Reader (PerkinElmer), to obtain the cell viability (%). As acontrol protein, His6-Dar(EGFR)-linker-TEV was used, which was obtainedfrom cleaved fusion proteins

The obtained results are demonstrated in FIG. 3 (HCT116) and FIG. 4(RKO). As shown in FIGS. 3 and 4, in both of HCT116 and RKO cell lines,the fusion proteins exhibit a decrease in cell viability by about 40˜60%at the concentration of 1.6 μM. Such effect on cell viability of acancer cell line is considerable, and at least about 10-fold superior tothat of other tumor suppressor (e.g., p53 n-terminal peptide, p18protein, etc.) when examined under the same conditions.

It should be understood that the exemplary embodiments described hereinshould be considered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A fusion protein comprising (1) granzyme B; or afragment of granzyme B comprising an active region of granzyme B and adipeptidyl peptidase I (DPPI) cleavage sequence or an auto-cleavagesequence, (2) a cell penetrating peptide, (3) a targeting moiety, and(4) a peptidase or protease cleavage site between the cell penetratingpeptide and the targeting moiety.
 2. The fusion protein of claim 1,wherein the cleavage sequence in the fragment of granzyme B comprisesfrom about 2 to about 20 amino acids, comprising the contiguous aminoacids GE or IEPD (SEQ ID NO: 36).
 3. The fusion protein of claim 1,wherein the cell penetrating peptide comprises one or more of: amembrane-translocation sequence (MTS) or a fragment thereof comprisingat least 5 contiguous amino acid residues within themembrane-translocation sequence, a macromolecule intracellulartransduction domain (MTD), TAT peptide, MTD103, TP10, Penetratin, modelamphipathic peptide (MAP), a cell penetrating fusion peptide comprisinga hydrophobic peptide and a basic peptide, or a combination thereof. 4.The fusion protein of claim 1, wherein the cell penetrating peptide is afusion peptide comprising: (1) a hydrophobic peptide comprising a totalof about 5 to about 40 amino acids, wherein the hydrophobic peptidecomprises about 70% or more hydrophobic amino acids based on the totalnumber of amino acids in the hydrophobic peptide, and wherein thehydrophobic amino acids are selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, methionine, proline,tryptophan, and phenylalanine; and (2) a basic peptide comprising abasic peptide unit, wherein the basic peptide unit comprises about 1 toabout 6 basic amino acids or a repeat comprising about 2 to about 6basic peptide units, wherein the basic amino acids are selected from thegroup consisting of lysine, arginine, and histidine.
 5. The fusionprotein of claim 4, wherein the hydrophobic peptide comprises SEQ ID NO:12 or a peptide fragment comprising 7 to 16 contiguous amino acidresidues of SEQ ID NO:
 12. 6. The fusion protein of claim 4, wherein thebasic peptide unit is KKKRK (SEQ ID NO: 15), KKKR(SEQ ID NO: 16), RKRK(SEQ ID NO: 17), RKRKRK (SEQ ID NO: 18), KKKKK (SEQ ID NO: 19),KKKKKR(SEQ ID NO: 20), KKKRKR(SEQ ID NO: 21), RRRRR(SEQ ID NO: 22), orRRRRRR(SEQ ID NO: 23).
 7. The fusion protein of claim 1, wherein thecleavage site of the peptidase or protease is a cleavage site recognizedby matrix metalloproteinase, cathepsin, urokinase-type plasminogenactivator, or a combination thereof.
 8. The fusion protein of claim 1,wherein the targeting moiety comprises an antibody, an antigen-bindingfragment of an antibody, a DARPin, an affibody, a lasso scaffold, acyclotide, a knottin, an avimer, a Kunitz domain, an anticalin, anadnectin, a pronectin, a fynomer, a nanofitin, an affilin, or acombination thereof, wherein the targeting moiety targets: a growthfactor a receptor tyrosine kinase protein, or a combination thereof. 9.The fusion protein of claim 1, wherein the fusion protein comprises inorder from N-terminus to C-terminus (1) the granzyme B or a fragmentthereof, (2) the cell penetrating peptide, (3) the cleavage site of apeptidase or protease, and (4) the targeting moiety.
 10. Apharmaceutical composition comprising the fusion protein of claim 1 anda carrier.
 11. A method of intracellular delivery of granzyme B,comprising administering the fusion protein of claim 1 to a subject inneed of intracellular delivery of granzyme B.
 12. A method of preventingor treating a cancer, comprising administering the fusion protein ofclaim 1 to a subject in need of preventing or treating a cancer.
 13. Amethod of improving a cell membrane penetrability of granzyme B,comprising preparing the fusion protein of claim
 1. 14. The fusionprotein of claim 8, wherein the growth factor comprises epidermal growthfactor (EGF), platelet-derived growth factor (PDGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), or acombination thereof; and the receptor tyrosine kinase protein comprisesan ErbB family member, insulin receptor, platelet-derived growth factorreceptor (PDGFR), fibroblast growth factor receptor (FGFR), vascularendothelial growth factor receptor (VEGFR), hepatocyte growth factorreceptor (HGFR), tropomyosin-receptor-kinase (Trk) receptor, Ephrin(Eph) receptor, AXL receptor, leukocyte receptor tyrosine kinase (LTK)receptor, TIE receptor, receptor tyrosine kinase-like orphan (ROR)receptor, discoidin domain receptor (DDR), RET receptor, KLG receptor,related to receptor tyrosine kinase (RYK) receptor, Muscle-SpecificKinase (MuSK) receptor, or a combination thereof.
 15. The fusion proteinof claim 1, wherein the fusion protein comprises SEQ ID NO: 1, 3, 5, 7,or
 9. 16. A polynucleotide encoding a fusion protein of claim
 1. 17. Amethod of preparing a fusion protein of claim 1 comprising expressing apolynucleotide encoding the fusion protein in a cell.